Probiotic sports nutrition compositions

ABSTRACT

The present application relates to nutritional compositions comprising lactic acid-producing bacteria.

RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. § 119(e)to U.S. Provisional Application No: 61/469,924, filed Mar. 31, 2011,which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present application relates to sports nutrition compositionscomprising lactic acid-producing bacteria.

BACKGROUND OF THE INVENTION

The gastrointestinal microflora plays a number of vital roles inmaintaining gastrointestinal tract function and overall physiologicalhealth. The growth and metabolism of the many individual bacterialspecies inhabiting the gastrointestinal tract depend upon the substratesavailable to them, most of which are derived from the diet.

Probiotic organisms are non-pathogenic, non-toxigenic microorganismsthat are beneficial to the host organism. Since probiotics do notgenerally permanently colonize the host, they are typically ingestedregularly for health promoting properties to persist.

SUMMARY OF THE INVENTION

The invention is based on the discovery that lactic acid-producingbacteria, particularly Bacillus coagulans, improve the absorption ofprotein compositions as well as micronutrient compositions. Themicroorganisms, e.g., spores remain viable, retain their beneficialprobiotic properties, and increase metabolism and/or absorption ofprotein in sports nutrition compositions. Unlike other probioticbacteria that die in the stomach or small intestine, the probioticorganisms described herein, e.g., Bacillus coagulans strain GBI-30 orBC³⁰, ATCC Designation Number PTA-6086, germinate in the stomach and/orsmall intestine. Accordingly, the invention describes probiotic sportsnutrition compositions. The invention provides an isolated Bacilluscoagulans bacterium (e.g., a spore) in sports nutrition compositions. Insome cases, the composition comprises at least 15% protein. For example,the composition comprises at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99% or more of a purified or processed protein (e.g., proteinextracted from a food-stuff) and a Bacillus coagulans spore.

The compositions described herein are suitable for consumption by amammal, e.g., any mammal, e.g., a human, a primate, a mouse, a rat, adog, a cat, a cow, a horse, or a pig. In a preferred embodiment, themammal is a human. For example, the invention includes a method ofenhancing lean muscle development or increasing lean body mass byadministering to a subject a composition comprising a Bacillus coagulansspore in a composition comprising at least 85% purified, processed,and/or isolated protein. The subject is a human being that desires toincrease muscle development, strength, or lean body mass, or an animal,e.g., livestock or performance animal such as a work animal or a racehorse, for which an increase in muscle development or strength isdesired. The compositions are also useful to confer clinical benefit toindividuals that are suffering from or at risk of developing a musclewasting condition, e.g., cachexia, as a result of disease such as canceror infection.

The invention provides a composition comprising a sports nutritioncomposition and an isolated Bacillus coagulans spore (e.g., in an amountof 1×10⁶ to 1×10¹⁴ colony forming units (CFU) of Bacillus coagulans perunit dose. For example, the isolated Bacillus coagulans comprise betweenabout 0.000001% to about 50% by weight of the composition, e.g., about1%, about 10%, about 20%, about 30%, about 40%, or about 50% by weightof the composition. For example, the isolated Bacillus coagulanscomprise between about 0.00001% and about 25% by weight of thecomposition, e.g., about 1%, about 2%, about 3%, about 4%, about 5%,about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%,about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about19%, about 20%, about 21%, about 22%, about 23%, about 24%, or about 25%by weight of the composition. In one example, one unit dose, e.g., oneserving, comprises one billion CFU Bacillus coagulans and 20-25 grams ofprotein (e.g., 23 grams of whey protein) in 30 grams of powder. Thebalance of the composition optionally comprises nutritionally inactiveingredients such as excipients, fillers, preservatives or nutritionallyactive ingredients such as vitamins or minerals.

The invention also provides bacterial species including Bacilluscoagulans, e.g., Bacillus coagulans hammer, preferably Bacilluscoagulans hammer strain Accession No. ATCC 31284, or one or more strainsderived from Bacillus coagulans hammer strain Accession No. ATCC 31284(e.g., ATCC Numbers: GBI-20, ATCC Designation Number PTA-6085; GBI-30 orBC³⁰, ATCC Designation Number PTA-6086; and GBI-40, ATCC DesignationNumber PTA-6087; see U.S. Pat. No. 6,849,256 to Farmer).

Optionally, the isolated Bacillus coagulans is in the form of a spore.Alternatively, the isolated Bacillus coagulans is in the form of avegetative cell. In another aspect, the isolated Bacillus coagulans isin the form of a mixture of vegetative cells and spores. The Bacilluscoagulans is predominantly in spore form, e.g., about 75%, about 80%,about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about99%, or about 100% spores. Alternatively, the Bacillus coagulans ispredominantly in vegetative form, e.g., about 75%, about 80%, about 85%,about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, orabout 100% vegetative cells.

Provided is an isolated Bacillus coagulans and a sports nutritioncomposition. In some cases, the compositions described herein comprise alarge amount of calories per unit dose to assist a subject in gainingweight, e.g., muscle weight or fat weight. A unit dose of thecompositions described herein is the amount of composition administeredto a consumer in a single dose, i.e., one serving. Unit-dose packagingis the packaging of a single dose, e.g., in a non-reusable container.For example, a unit dose refers to a physically discrete unit suitableas unitary dosages for an individual, each unit containing apredetermined quantity of active material calculated to produce thedesired therapeutic effect, in association with a suitablepharmaceutical carrier, diluent, or excipient. Suitable packagingincludes single or multiple unit dosages.

Compositions that provide a large amount of calories to assist a subjectin gaining weight are referred to as “weight gainers.” In these cases,the composition comprises between about 100 and about 10,000 foodCalories (kcal) per unit dose (i.e., serving), e.g., between 250 and5,000 kcal, between 500 and 3,000 kcal, between 750 and 2,500 kcal, orbetween 1,000 and 2,000 kcal, e.g., about 1,000 kcal, about 1,100 kcal,about 1,200 kcal, about 1,300 kcal, about 1,400 kcal, about 1,500 kcal,about 1,600 kcal, about 1,700 kcal, about 1,800 kcal, about 1,900 kcal,or about 2,000 kcal. One exemplary amount of calories is 1,230 kcal.Alternatively, the compositions do not comprise a large amount ofcalories. For example, the compositions comprise between about 10 and500 kcal, e.g., between about 20 and 250 kcal, between about 50 and 200kcal, or between about 100 and 150 kcal, e.g., about 100 kcal, about 110kcal, about 120 kcal, about 130 kcal, about 140 kcal, or about 150 kcal.One exemplary amount of calories is about 150 kcal.

Preferably, the composition comprises protein. For example, the proteincomprises about 1% to about 99% by weight of the composition, e.g.,about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% byweight of the composition. For example, the composition comprisesbetween 1 gram and 500 grams of protein, e.g., about 10 grams, about 15grams, about 20 grams, about 25 grams, about 30 grams, about 35 grams,about 40 grams, about 45 grams, about 50 grams, about 55 grams, about 60grams, about 65 grams, about 70 grams, about 75 grams, about 80 grams,about 85 grams, about 90 grams, about 95 grams, about 100 grams, about150 grams, about 200 grams, about 250 grams, about 300 grams, about 350grams, about 400 grams, about 450 grams, or about 500 grams of protein.One exemplary composition comprises about 62% protein. For example, thecomposition comprises about 39 total grams, of which about 24 grams isprotein. Another exemplary composition comprises about 65% protein. Forexample, the composition comprises about 31 total grams, of which about20 grams is protein. Another exemplary composition comprises about 15%protein. For example, the composition comprises about 325 total grams,of which about 50 grams is protein. A typical protein compositioncomprises about 50-100% protein such as whey protein. For example, aunit dose comprises 1 billion CFU of Bacillus coagulans and 23 grams ofwhey protein in a total of 30 grams of powder.

In one aspect, the sports nutrition composition comprises purified orprocessed protein, such as soy protein, whey protein, rice protein, hempseed protein, casein protein or milk protein. Preferably, the proteincomprises whey protein. The protein comprises an amino acid selectedfrom the group consisting of isoleucine, alanine, leucine, arginine,lysine, aspartate, aspartic acid, methionine, cysteine, phenylalanine,threonine, tryptophan, glycine, valine, proline, histidine, serine,tyrosine, asparagine, selenocysteine, pyrrolysine, glutamate, glutamicacid, and glutamine. Optionally, the sports nutrition compositioncomprises an isolated Bacillus coagulans and protein, and furthercomprises creatine, calcium, sodium caseinate, whey peptides, orlactoferrin. Alternatively, the invention provides an isolated Bacilluscoagulans and a sports nutrition composition selected from the groupconsisting of creatine, calcium, sodium caseinate, whey peptides, andlactoferrin.

The compositions optionally further comprise additional ingredientsselected from the group consisting of sodium, potassium, sugar,carbohydrates, dietary fiber, vitamin A, vitamin C, calcium, iron,vitamin D, vitamin E, thiamin, riboflavin, niacin, vitamin B6, folate,vitamin B12, biotin, pantothenic acid, phosphorus, iodine, magnesium,zinc, selenium, copper, manganese, chromium, and molybdenum.

An exemplary composition comprises glucose polymer, a protein blend(i.e., whey protein concentrate, whey protein isolate, egg albumin, milkprotein isolate, and partially hydrolyzed whey protein), rice proteinconcentrate, brown rice concentrate, taurine, L-glutamine, non-dairycreamer (i.e., sunflower oil, corn syrup solids, sodium caseinate,mono-and diglycerides, dipotassium phosphate, tricalcium phosphate, soylecithin, and tocopherols), natural and artificial flavors, xantham gum,calcium citrate, potassium citrate, dipotassium phosphate, cellulosegum, tricalcium phosphate, magnesium aspartate, rice starch,carrageenan, vitamin-mineral blend (i.e., ascorbic acid, niacinamide,d-Alpha tocopheryl succinate, d-calcium pantothenate, zinc citrate,pyridoxine hydrochloride, ferrous fumarate, thiamine mononitrate,riboflavin, manganese amino acid chelate, beta-carotene, coppergluconate, folic acid, biotin, potassium iodide, chromiumpolynicotinate, molybdenum amino acid chelate, selenomethionine,cyanocobalamin, and cholecalciferol), GANEDEN BC-30™ (Bacillus coagulansGBI-30, ATCC Designation Number PTA-6086), sucralose, acesulfamepotassium, and lactase.

Another exemplary composition comprises the following ingredients: wheyprotein concentrate, brown rice protein concentrate, whey proteinisolate, egg albumin, milk protein isolate, partially hydrolyzed wheyprotein, glucose polymer, taurine, L-glutamine, nondairy creamer (i.e.,sunflower oil, corn syrup solids, sodium caseinate, mono- anddiglycerides, dipotassium phosphate, tricalcium phosphate, soy lecithin,and tocopherols), dicalcium phosphate, natural and artificial flavors,xanthan gum, cellulose gum, carrageenan, lecithin, acesulfame potassium,sucralose, lactase, and GANEDEN BC-30™ (Bacillus coagulans GBI-30, ATCCDesignation Number PTA-6086).

The compositions contain active ingredients, e.g., protein and GANEDENBC-30™ (Bacillus coagulans GBI-30, ATCC Designation Number PTA-6086),and inactive ingredients, e.g., excipients, binders, or fillers. Fillersfill out the size of the compositions, making it practical to produceand convenient for the consumer to use. By increasing the bulk volume,the fillers make it possible for the final product to have the propervolume for consumer handling. Suitable fillers include xantham gum,cellulose gum, lecithin, lactose, sucrose, glucose, mannitol, sorbitol,calcium carbonate, and magnesium stearate.

In some cases, the compositions do not comprise certain ingredients. Forexample, the composition does not include a sugar (e.g., glucose,fructose, galactose, maltose or lactose), gluten, aspartame, and/orartificial coloring.

Exemplary forms for the compositions described herein include a proteinpowder, a ready to drink protein shake, a protein bar, a protein bite,and a protein gel.

The invention provides compositions comprising a dry mix for sportsnutrition compositions comprising an isolated Bacillus coagulansbacterium and a sports nutrition composition. The dry mix is between0.01% and 50% Bacillus coagulans bacterium, e.g., about 1%, about 5%,about 10%, about 15%, about 20%, about 25%, about 35%, about 45%, orabout 50% Bacillus coagulans bacterium. In some cases, the dry mix isabout 15% Bacillus coagulans bacterium. For example, about 100 pounds ofdry mix contains about 15 pounds of Bacillus coagulans bacterium andabout 85 pounds of sports nutrition composition. Optionally, theisolated Bacillus coagulans is in the form of a spore. Alternatively,the isolated Bacillus coagulans is in the form of a vegetative cell. Inanother aspect, the isolated Bacillus coagulans is in the form of amixture of vegetative cells and spores. Preferably, the isolatedBacillus coagulans is in the form of a spore. More preferably, thebacterium is present as at least 80% spores, e.g., at least 85%, atleast 90%, at least 95%, at least 98%, or at least 99% spores.

Bacterial species suitable for use in the methods describe hereininclude Bacillus coagulans, e.g., Bacillus coagulans hammer, preferablyBacillus coagulans hammer strain Accession No. ATCC 31284, or one ormore strains derived from Bacillus coagulans hammer strain Accession No.ATCC 31284 (e.g., ATCC Numbers: GBI-20, ATCC Designation NumberPTA-6085; GBI-30 or GANEDEN BC-30™, ATCC Designation Number PTA-6086;and GBI-40, ATCC Designation Number PTA-6087; see U.S. Pat. No.6,849,256 to Farmer).

In some cases, the dry mix also includes a protein selected from thegroup consisting of soy protein, whey protein, rice protein, hemp seedprotein, and casein protein. In other cases, the sports nutritioncomposition also includes creatine, calcium, sodium caseinate, wheypeptides, and lactoferrin.

Methods of enhancing lean muscle development, recovery, or repair arecarried out by administering to a subject desiring an enhancement of thelean muscle development, recovery or repair the compositions describedherein, e.g., a composition comprising a sports nutrition compositionand an isolated Bacillus coagulans spore, wherein the compositioncomprises at least 80% protein. The composition is optionallyadministered prior to or after an exercise period. For example, thecomposition is administered within 60 minutes of an exercise period,e.g., within 15 minutes, within 30 minutes, or within 45 minutes.Alternatively, the composition is administered within 2 hours, within 5hours, or within 8 hours of an exercise period.

The absorption of the protein is increased in the subject after theadministration of Bacillus coagulans in combination with protein ascompared to the absorption of the protein after the administration ofprotein in the absence of Bacillus coagulans. For example, the rate ofabsorption (t_(MAx)) or overall absorption (from area under the curve(AUC)_(0-4h) and C_(MAX)) are increased after the administration ofBacillus coagulans in combination with protein as compared to after theadministration of protein in the absence of Bacillus coagulans , asmeasured by changes in amino acid levels in, e.g., blood over a one hourtest period, a two hour test period, a four hour test period, an eighthour test period, a twelve hour test period, or a twenty-four hour testperiod. For example, protein absorption is increased by at least 10%,50%, 2-fold, 5-fold, 10-fold, or more following administration ofprotein and Bacillus coagulans compared to protein without Bacilluscoagulans.

The absorption of a vitamin is increased in the subject after theadministration of Bacillus coagulans in combination with protein ascompared to the absorption of the vitamin after the administration ofprotein in the absence of Bacillus coagulans. Exemplary vitamins includevitamin A, vitamin B₁, vitamin B₂, vitamin B₃, vitamin B₅, vitamin B₆,vitamin B₇, vitamin B₉, vitamin B₁₂, or vitamin C, vitamin D, vitamin E,and vitamin K₁.

In other cases, the absorption of a chemical element is increased in thesubject after the administration of Bacillus coagulans in combinationwith protein as compared to the absorption of the chemical element afterthe administration of protein in the absence of Bacillus coagulans.Exemplary chemical elements include sodium, potassium, calcium, iron,and zinc.

Optionally, the Bacillus coagulans aids in the digestion of the sportsnutrition composition, e.g., protein. For example, abdominal bloating,intestinal pain, passage of gas, loose bowel movements, and excessiveabdominal gurgling are reduced following consumption of protein andBacillus coagulans compared to the consumption of protein withoutBacillus coagulans. However, subjects ingesting the product preferablyhave not been diagnosed with gastrointestinal disease or an inflammatorybowel condition.

The compositions are also useful in the prophylactic or therapeutictreatment of conditions associated with gastrointestinal infection byvarious pathogens, thereby supporting digestive health. The probioticsports nutrition compositions of the invention are also used in themethods described herein for boosting the immune system. In some cases,the combination of Bacillus coagulans and protein works synergisticallyresulting in an enhanced inhibition of pathogens in the gastrointestinaltract of a subject, as compared to the administration of either Bacilluscoagulans or protein alone. In other cases, the combination of Bacilluscoagulans and protein works synergistically resulting in an enhancedboost of the immune system, as compared to the administration of eitherBacillus coagulans or protein alone.

Optionally, the compositions of the invention additionally compriseL-alanine and/or inosine to promote the germination of the spores in thestomach and/or small intestine.

A probiotic lactic acid-producing bacteria suitable for use in themethods and compositions of the invention produces acid and isnon-pathogenic. Purified and/or isolated Bacillus coagulans isparticularly useful as a probiotic in the compositions described herein.By “purified” or “substantially purified” is meant a Bacillus coagulansbacterium that is substantially free of contaminating microorganisms orother macromolecules, e.g., polysaccharides, nucleic acids, or proteins.

Purified defines a degree of sterility that is safe for administrationto a human subject, e.g., lacking infectious or toxic agents.Specifically, as used herein, an “isolated” or “purified” Bacilluscoagulans or protein is substantially free of other cellular material,culture medium, chemical precursors, or other chemicals when chemicallysynthesized. Purified compounds are at least 60% by weight (dry weight)the compound of interest. Preferably, the preparation is at least 75%,more preferably at least 90%, and most preferably at least 99%, byweight the compound of interest. Purity is measured by any appropriatestandard method, for example, by column chromatography, polyacrylamidegel electrophoresis, or HPLC analysis.

The Bacillus coagulans Hammer strains of the invention arenon-pathogenic and generally regarded as safe for use in human nutrition(i.e., GRAS classification) by the U.S. Federal Drug Administration(FDA) and the U.S. Department of Agriculture (USDA), and by thoseskilled in the art. Furthermore, the Bacillus coagulans Hammer strainsof the invention germinate at or below human body temperature, renderingthem useful as probiotics. Many Bacillus coagulans strains outside theHammer group have mostly industrial applications, little or nonutritional benefit, and environmental contaminants that have not beenevaluated for safety. Moreover, many other non-Hammer strains ofBacillus coagulans grow optimally at temperatures that exceed human bodytemperature and, thus, do not germinate efficiently in the human body.Such strains are less or not suitable as probiotics for humanconsumption.

The transitional term “comprising,” which is synonymous with“including,” “containing,” or “characterized by,” is inclusive oropen-ended and does not exclude additional, unrecited elements or methodsteps. By contrast, the transitional phrase “consisting of” excludes anyelement, step, or ingredient not specified in the claim. Thetransitional phrase “consisting essentially of” limits the scope of aclaim to the specified materials or steps “and those that do notmaterially affect the basic and novel characteristic(s)” of the claimedinvention.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims. Unless otherwise defined, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention,suitable methods and materials are described below. All publications,patent applications, patents, Genbank/NCBI accession numbers, and otherreferences mentioned herein are incorporated by reference in theirentirety. In the case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

DETAILED DESCRIPTION OF THE INVENTION

The probiotic organisms of the invention are non-pathogenic,non-toxigenic, retain viability during storage, and survive passagethrough the stomach and small intestine. Non-pathogenic lacticacid-producing bacteria (i.e., “lactic acid bacteria”), such as theexemplary Bacillus coagulans, remain viable and retain their beneficialprobiotic properties in sports nutrition compositions. Prior to theinvention described herein, probiotics were very susceptible to harshenvironment of the stomach and small intestine. Unlike other probioticsthat die in the stomach or small intestine, the probiotic organismsdescribed herein, e.g., Bacillus coagulans strain GBI-30 or GANEDENBC-30™, ATCC Designation Number PTA-6086, germinate in the stomachand/or small intestine. Specifically, the probiotic spores describedherein can survive passage through the stomach and small intestine.

Probiotic Lactic Acid-Producing Bacteria

The sports nutrition compositions include a lactic acid-producingbacterium, such as a spore-forming Bacillus species, e.g., B. coagulans.Preferably, the spore-forming Bacillus species of the invention is B.coagulans Hammer. There are many suitable bacteria identified asdescribed herein, although the invention is not limited to currentlyknown bacterial species insofar as the purposes and objectives of thebacteria is described. The property of acid production is important tothe effectiveness of the probiotic lactic acid-producing bacteria ofthis invention.

Exemplary methods and compositions are described herein using Bacilluscoagulans as a probiotic. Purified and/or isolated Bacillus coagulans isparticularly useful as a probiotic in sports nutrition compositions.Probiotic B. coagulans is non-pathogenic and is generally regarded assafe (i.e., GRAS classification) by the U.S. Federal Drug Administration(FDA) and the U.S. Department of Agriculture (USD A), and by thoseskilled in the art.

Bacillus coagulans is a non-pathogenic gram positive spore-formingbacteria that produces L(+) lactic acid (dextrorotatory) in fermentationconditions. It has been isolated from natural sources, such asheat-treated soil samples inoculated into nutrient medium (Bergey'sManual off Systemic Bacteriology, Vol. 2, Sneath, P.H.A., et al, eds.,Williams & Wilkins, Baltimore, Md., 1986). Purified B. coagulans strainshave served as a source of enzymes including endonucleases (e.g., U.S.Pat. No. 5,200,336), amylase (U.S. Pat. No. 4,980,180), lactase (U.S.Pat. No. 4,323,651), and cyclo-malto-dextrin glucano-transferase (U.S.Pat. No. 5,102,800). B. coagulans has been used to produce lactic acid(U.S. Pat. No. 5,079,164). A strain of B. coagulans (referred to as L.sporogenes Sakaguti & Nakayama (ATCC 31284)) has been combined withother lactic acid producing bacteria and B. natto to produce a fermentedfood product from steamed soybeans (U.S. Pat. No. 4,110,477).

Bacterial species include Bacillus coagulans , e.g., Bacillus coagulanshammer, preferably Bacillus coagulans hammer strain Accession No. ATCC31284, or one or more strains derived from Bacillus coagulans hammerstrain Accession No. ATCC 31284 (e.g., ATCC Numbers: GBI-20, ATCCDesignation Number PTA-6085; GBI-30 (GANEDEN BC-30™), ATCC DesignationNumber PTA-6086; and GBI-40, ATCC Designation Number PTA-6087; see U.S.Pat. No. 6,849,256 to Farmer).

Bacillus coagulans was previously mis-characterized as a Lactobacillusand labeled as Lactobacillus sporogenes. However, initial classificationwas incorrect because Bacillus coagulans produces spores and excretesL(+)-lactic acid through metabolism. Both of these characteristicsprovide key features to the utility of Bacillus coagulans. Thesedevelopmental and metabolic aspects required that the bacterium beclassified as a lactic acid Bacillus. In addition, it is not generallyappreciated that classic Lactobacillus species are unsuitable forcolonization of the gut due to their instability in the harsh (i.e.,acidic) pH environment of the bile, particularly human bile. Bycontrast, Bacillus coagulans ATCC Designation Number PTA-6085; GBI-30(GANEDEN BC-30™) is able to survive and colonize the gastrointestinaltract in the bile environment and even grow in this low pH range.

Probiotic Activity of Bacillus Coagulans

It is well-documented clinically that many species of bacterial, mycoticand yeast pathogens possess the ability to cause a variety ofgastrointestinal disorders including, but not limited to disruption ofnormal gastrointestinal biochemical function, necrosis ofgastrointestinal tissues, and disruption of the bioabsorption ofnutrients, and like conditions. The probiotic microorganism-containingcompositions described herein inhibit these pathogens. In the presentcase, subjects are generally healthy, e.g., they have not been diagnosedwith a gastrointestinal disease (e.g., irritable bowel syndrome orCrohn's disease), an inflammatory bowel condition, or an autoimmunedisease. The compositions and methods described herein are useful toincrease absorption of protein and/or macro nutrients in a nutritionalcomposition.

In one aspect, a Bacillus coagulans strain is included in thecomposition in the form of vegetative cells. In another aspect, theBacillus coagulans strain is included in the composition in the form ofspores. The invention also provides for including the Bacillus coagulansstrain in the composition in the form of a powder, a dried cell mass, astabilized paste, or a stabilized gel.

Because Bacillus spores are heat and pressure-resistant and can bestored as a dry powder, they are particularly useful for formulationinto and manufacture of products such as the various sports nutritioncompositions described herein. Specifically, the probiotic organismsdescribed herein, e.g., Bacillus coagulans strain GBI-30 or GANEDENBC-30™, ATCC Designation Number PTA-6086, survive passage through thestomach and small intestine. A Bacillus species is well suited for thepresent invention, particularly species having the ability to formspores which are relatively resistant to heat and other conditions,making them ideal for storage (shelf-life) in product formulations,e.g., sports nutrition compositions. Due to the shelf-stable propertiesof the Bacillus coagulans strains described herein, e.g.. Bacilluscoagulans strain GBI-30 or GANEDEN BC-30™, ATCC Designation NumberPTA-6086, the product formulations of the invention are not confined toa refrigerator and may be stored at room temperature. The Bacilluscoagulans of the invention survives storage (shelf-life) from about 12days to about 2 years; from about 1 month to about 18 months; from about3 months to about 1 year; or from about 6 months to about 9 months.

The probiotic organisms described herein, e.g., Bacillus coagulansstrain GBI-30 or GANEDEN BC-30™, ATCC Designation Number PTA-6086,promote digestive and oral health and support the immune system. Theability of Bacillus coagulans to inhibit various bacterial pathogens wasquantitatively ascertained by use of an in vitro assay. This assay ispart of a standardized bacterial pathogen screen (developed by the U.S.Food and Drug Administration(FDA)) and is commercially available onsolid support disks (DIFCO® BACTROL® Antibiotic Disks). To perform theassay, potato-dextrose plates (DIFCO®) were initially prepared usingstandard procedures. The plates were then individually inoculated withthe bacteria (approximately 1.5×10⁶ CFU) to be tested so as to form aconfluent bacterial bed.

Inhibition of microorganisms (e.g. gastrointestinal pathogens) byBacillus coagulans was subsequently ascertained by placing approximately1.8×10⁶ CFU of Bacillus coagulans in 10 μl of broth or buffer, directlyin the center of the potato-dextrose plate with one test locus beingapproximately 8 mm in diameter per plate. A minimum of three test lociwere used for each assay. The negative control consisted of a 10 μlvolume of a sterile saline solution, whereas the positive controlconsisted of a 1 μl volume of glutaraldehyde. The plates were thenincubated for approximately about 18 hr at 30° C., at which time thezones of inhibition were measured. As designated herein, “excellentinhibition” means the zone was 10 mm or greater in diameter; and “goodinhibition” means the zone was greater than 2 mm in diameter but lessthan 10 mm in diameter.

As expected, no “inhibition” was seen with the negative, saline control,and excellent “inhibition” (approximately 16.2 mm diameter; average ofthree tests) was seen with the positive, glutaraldehyde control. For theenteric microorganisms tested, the following inhibition by Bacilluscoagulans was found: (\) Clostridium species—excellent inhibition; (ii)Escherichia coli—excellent inhibition; (in) Clostridiumspecies—excellent inhibition, where the zone of inhibition wasconsistently greater than 15 mm in diameter. Similarly, excellentinhibition was also seen for the opportunistic pathogens Pseudomonasaeruginosa, and Staphylococcus aureus. Pathogenic enteric bacteria whichwere inhibited by Bacillus coagulans activity include, but are notlimited to: Staphylococcus aureus; Staphylococcus epidermidis;Streptococcus pyogenes; Pseudomonas aeruginosa; Escherichia coli(enterohemorragic species); numerous Clostridium species (e.g.,Clostridium perfingens; Clostridium botulinum; Clostridium tributrycum;Clostridium sporogenes, and the like); Gardnereia vaginalis;Proponbacterium aenes; Aeromonas hydrophia; Aspergillus species; Proteusspecies; and Klebsiella species.

Micro-Encapsulation

In one aspect, the lactic-acid producing bacteria are incorporated intoa microcapsule coating prior to addition to the sports nutritioncomposition, using any micro-encapsulation process well-known in theart. The isolated Bacillus coagulans are packaged, or encapsulated,within another material in order to protect the bacteria from thesurrounding environment. The capsules of the invention range in sizefrom one-thousandth of a millimeter to seven millimeters. The internalingredients of the microcapsule are released from their shells invarious ways, including mechanical rupture of the capsule wall,dissolution of the wall, melting of the wall and diffusion through thewall. Thus, micro-encapsulation provides additional protection to theisolated Bacillus bacterium during manufacturing and storage of thesports nutrition compositions of the invention. Physical methods ofmicro-encapsulation include pan coating, air-suspension coating,centrifugal extrusion, vibrational nozzle, and spray-drying. Chemicalmethods of micro-encapsulation include interfacial polymerization,in-situ polymerization, and matrix polymerization.

Alternatively, the lactic-acid producing bacteria is added to the sportsnutrition composition without micro-encapsulation.

Probiotic Sports Nutrition Compositions

The invention includes a method of increasing protein absorption,enhancing lean muscle development, and/or increasing lean body mass byadministering to a subject a composition comprising a Bacillus coagulansspore in a composition comprising at least 50% purified or processedprotein, e.g., a composition that contains at least 75% whey protein orother protein source. The sports nutrition compositions are suitable forhuman or animal consumption. A typical subject is a healthy adult, e.g.,greater than 16 years old. Alternatively, the sports nutritioncompositions may also be administered to children under 18 years of age,e.g., under 15 years of age, under 10 years of age, or under 5 years ofage. Alternatively, the sports nutrition compositions are administeredto children and adults of all ages. The subject is a human being thatdesires to increase muscle development, strength or lean body mass, oran animal, e.g., livestock or performance animal such as a work animalor a race horse, for which an increase in muscle development or strengthis desired. The compositions are also useful to confer clinical benefitto individuals that are suffering from or at risk of developing a musclewasting condition, e.g., cachexia, as a result of disease such as canceror infection.

Sports nutrition compositions are prepared by mixing dry powderingredients (e.g., protein and Bacillus coagulans). Sports nutritioncompositions include bodybuilding or body maintenance foods, e.g.,weight gainers, commonly used by those involved in bodybuilding andathletics. They include protein (the most widely used such sportsnutrition composition), amino acids, glutamine, essential fatty acids,meal replacement products, prohormones, creatine, thermogenic products,testosterone boosters, and branched-chain amino acids (BCAA). The threebranched-chain amino acids (BCAAs) include leucine, isoleucine, andvaline. Unlike other amino acids, BCAAs are metabolized in the muscleand have an anabolic/anti-catabolic effects. BCAAs account for 33% ofmuscle protein. Amino acids are the building blocks of protein, whichthe body metabolizes in the stomach and intestines. Protein powdercontains amino acids such as isoleucine, alanine, leucine, arginine,lysine, aspartate, aspartic acid, methionine, cysteine, phenylalanine,threonine, tryptophan, glycine, valine, proline, histidine, serine,tyrosine, asparagine, selenocysteine, pyrrolysine, glutamate, glutamicacid, and glutamine.

Glutamine is the most abundant amino acid found in human muscle;however, the body's natural glutamine stores are depleted duringanaerobic exercise. Thus, the sports nutrition compositions of theinvention optionally include glutamine. Serum glutamine is used by thebody to counteract acidosis resulting from exercise. In order toreplenish the loss of glutamine from the bloodstream, the bodycatabolizes glutamine from the muscle. Thus, ingestion of glutaminecombats muscle tissue wasting.

Suitable types of protein include soy protein, whey protein, riceprotein, hemp protein, casein protein, and egg-white protein (e.g., eggalbumin). Soy protein isolated from soybeans contains isoflavones, atype of phytoestrogen. Whey protein contains high levels of all theessential amino acids and branched-chain amino acids. For example, theamino acids regarded as essential for humans are phenylalanine, valine,threonine, tryptophan, isoleucine, methionine, leucine, lysine, andhistidine. Whey protein also has the highest content of the amino acidcysteine, which aids in the biosynthesis of glutathione. For thisreason, whey protein provides amino acids to aid in muscle recovery.Whey protein is derived from the process of making cheese from milk.There are two types of whey protein: whey concentrate (29%-89% proteinby weight) and whey isolate (90%+protein by weight). Another type ofprotein includes rice protein, which is made from whole grain, acomplete protein source that is highly digestible and allergen free.Hemp protein from hemp seed contains complete and highly-digestibleprotein. Hemp oil is high in essential fatty acids, i.e.,alpha-linolenic acid (an omega-3 fatty acid) and linoleic acid (anomega-6 fatty acid). Casein protein (or milk protein) has glutamine, andcasomorphin. Finally, egg-white protein is a lactose- and dairy-freeprotein.

Proteins come in various forms, including protein powder, ready to drinkprotein shakes, bars, bites, oats, and gels. Protein powders areavailable in a wide variety of flavors including pineapple, orange,fruit punch, mixed berry, mango, cookies and cream, strawberry,strawberry banana, French vanilla, vanilla, vanilla ice cream, vanillamilkshake, banana, banana cream, Dutch chocolate, mocha cappuccino,double rich chocolate, chocolate caramel, chocolate milkshake, extrememilk chocolate, chocolate mint, chocolate chip, and chocolate. Theprotein powder is mixed with water, milk or juice (e.g., grapefruitjuice, grape juice, orange juice, etc.), and often flavoring, resultingin a form known as a “protein shake” (as in milkshake) or “pudding”.Protein powder is generally consumed immediately before or afterexercising, or in place of a meal. The theory behind this regimen isthat having a sufficient protein intake allows for efficient growth andrepair of muscle tissue. Protein powders also aid in fat loss, musclebuilding, and slow the aging process.

Meal replacement products (MRPs) are either pre-packaged powdered drinkmixes or edible bars designed to replace prepared meals. MRPs aregenerally high in protein, low in fat, have a low to moderate amount ofcarbohydrates, and contain a wide array of vitamins and minerals. Themajority of MRPs use whey protein, casein (often listed as calciumcaseinate or micellar casein), soy protein, and/or egg albumin asprotein sources. Carbohydrates are typically derived from maltodextrin,oat fiber, brown rice, and/or wheat flour. Some MRPs also contain flaxseed oil powder as a source of essential fatty acids.

Sports nutrition compositions may also contain other additionalingredients that are beneficial for bodybuilding, including calcium,sodium caseinate, whey peptide, glutamine peptides, L-glutamine, calciumalpha-ketoglutarate, additional amino acids, lactoferrin, conjugatedlinoleic acid, medium chain triglycerides, and creatine (e.g., creatinemonohydrate). Creatine is a nitrogenous organic acid that is found inthe muscle tissue of vertebrates mainly in the form of phosphocreatinereplenishment of ATP, and supplies energy for muscle contraction.Creatine improves strength, energy, muscle mass, recovery times, brainfunction, and reduces mental fatigue. Creatine is available in a varietyof forms, including creatine monohydrate and creatine ethyl ester.

Although it is known that athletes and bodybuilders may need anincreased intake of protein, the exact amount is highly individualizedand dependent on the type and duration of the exercise as well as thephysiological make up of the individual. Research by Tarnopolsky et al.(1988) showed that for bodybuilding individuals, 1.97 g of protein perkg of body weight per day is recommended, whereas endurance athletesrequire 1.37 g/kg/d of protein. Studies suggest that there are differentprotein requirements for anaerobic and aerobic exercise. Enduranceathletes in aerobic activity may have increased daily protein intake at1.2-1.4 g per kg body weight per day, whereas strength training athletesperforming anaerobic activity may have increased daily protein intakeneeds at 1.4-1.8 g per kg body weight so as to enhance muscle proteinsynthesis or to make up for the loss of amino acid oxidation duringexercise.

Preferably, the sports nutrition composition ingredients are blendedtogether as dry ingredients. Exemplary ingredients of the sportsnutrition compositions described herein include protein and Bacilluscoagulans. An exemplary strain of Bacillus coagulans suitable in thesports nutrition compositions described herein includes GBI-30 orGANEDEN BC-30™, ATCC Designation Number PTA-6086.

The dry mix for sports nutrition compositions comprises an isolatedBacillus coagulans bacterium. The dry mix is approximately 1 billion CFUof Bacillus coagulans bacterium per unit dose. Optionally, the dry mixis about 15% Bacillus coagulans bacterium and 85% protein. For example,about 100 pounds of dry mix contains about 15 pounds of Bacilluscoagulans bacterium and about 85 pounds of protein. The dry mix isbetween about 1% and about 50% by weight of the sports nutritioncomposition, e.g., about 1% to about 20%, about 5% to about 15%; about6%, about 7%, about 8%, about 9%, or about 10% by weight of the sportsnutrition composition. For example, a 3 gram sports nutritioncomposition contains about 7% dry mix by weight of the sports nutritioncomposition. A 3.8 to 4 gram sports nutrition composition contains about8-9% dry mix by weight of the sports nutrition composition.

As the recommended dietary allowances (RDA or recommended daily intake;RDI) is about 1×10⁹ bacterium (according to EU guidelines), preferably,the sports nutrition composition comprises at least about 1×10⁹ viablebacteria. In another aspect, the sports nutrition composition comprisesat least about 1×10⁶ to 1×10⁷; at least about 1×10⁷ to 1×10⁸; or atleast about 1×10⁸ to 1×10⁹ viable bacteria.

The Bacillus and/or Bacillus coagulans in the form of a dried sporecomposition is mixed with dry whey powder or is applied using any of avariety of known methods including, for example, applying a powder,spray-drying the probiotic onto the sports nutrition composition, orsoaking the composition in a solution containing the probiotic. Any of avariety of methods for placing the bacterial composition into a sportsnutrition composition can be used. In one aspect, a “spray-dry” methodis used, in which the compositions are exposed in a low humidity chamberto an atomized mix containing a liquid composition, where the chamber issubsequently exposed to approximately 80-110° F. to dry the liquid,thereby impregnating the material of the sports nutrition compositionwith the components.

In some cases, Bacillus coagulans bacteria in the form of a spray-driedpowder is included in or on the surface of the sports nutritioncompositions described herein. Preferably, the isolated Bacilluscoagulans is in the form of a spore, as the Bacillus coagulans spores ofthe invention are protected by a hardened coating that can withstandgastric acid and bile salts for delivery to the small and largeintestines. The isolated Bacillus coagulans are at least 85%, at least90%, at least 95%, or at least 99% pure spores. Alternatively, theisolated Bacillus coagulans is in the form of a vegetative cell. In oneaspect, the isolated Bacillus coagulans are at least 85%, at least 90%,or at least 95% pure vegetative cells. In another aspect, the isolatedBacillus coagulans is in the form of a mixture of vegetative cells andspores. The Bacillus coagulans mixture is 90% spores, 10% vegetativecells; 75% spores, 25% vegetative cells; 60% spores, 40% vegetativecells; 50% spores, 50% vegetative cells; 60% vegetative cells, 40%spores; 75% vegetative cells; 25% spores; or 90% vegetative cells, 10%spores.

In one aspect, the amount of bacteria is about 10⁴ to 10¹⁴ colonyforming units (CFU) of bacteria per gram of probiotic composition (i.e.,vegetative cells and/or bacterial spores), preferably 10⁵ to 10¹³ CFU/gof sports nutrition composition. Alternatively, the concentrations are10⁸ to 10¹³ CFU/g; 10⁹ to 10¹² CFU/g; or 10¹⁰ to 10¹¹ CFU/g of sportsnutrition composition. In one aspect, the amount of bacteria is about1×10⁶ CFU per gram of sports nutrition composition. The actual amount ina sports nutrition composition will vary. A typical concentration isfrom approximately 1×10⁷ to 1×10¹² CFU; 1×10⁸ to 1×10¹¹ CFU; or 1×10⁹ to1×10¹⁰ CFU of viable bacterium or spores/g of sports nutritioncomposition.

Following drying, the sports nutrition composition is ready forimmediate use or for storage in a sterile package, e.g., a 3-ouncepackage (e.g., a bag or a bottle), a 6-ounce package, a 9-ounce package,a 12-ounce package, a 15-ounce package, an 18-ounce package, a 24-ouncepackage, a 48-ounce package, 80-ounce package, or 100-ounce package. Inanother example, the dried powder is packaged in unit dose quantities,e.g., 5 grams, 10 grams, 20 grams, 30 grams, 40 grams, 50 grams, 60grams, 70 grams, 80 grams, 90 grams, or 100 gram packets. Alternatively,the dried powder is packaged in bulk, e.g., about 500 grams, about 600grams, about 700 grams, about 800 grams, about 900 grams, about 1,000grams, about 1,250 grams, about 1,500 grams, about 1,750 grams, about2,000 grams, about 2,250 grams, about 2,500 gram, or about 3,000 gramcontainers. In one aspect, the invention provides for storing the sportsnutrition composition in a sterile package at room temperature prior toconsumption. Alternatively, the composition is consumed immediately.

The active ingredients (i.e., Bacillus coagulans and protein), comprisebetween about 0.01% to about 10%; 0.01% to about 1%; or about 0.05% toabout 0.1% by weight of the probiotic sports nutrition composition.Optionally, the isolated Bacillus coagulans comprise about 1 mg to about10 g; about 10 mg to about 1 g; or about 25 mg to about 75 mg by weightof the probiotic composition. Most preferably, the amount of Bacilluscoagulans bacteria is about 5×10⁷ colony forming units (CFU) of bacteriaper gram of food matrix.

In some cases, the Bacillus coagulans spores are incorporated into anytype of dry or lyophilized product which is dissolved or mixed withwater, milk, or juice, so long as the temperature of the Bacilluscoagulans spore-containing mixture is raised to the required heat-shocktemperature (i.e., 80° C. for 5 minutes) necessary for germination ofthe spores. The Bacillus coagulans spores may either be incorporatedinto the dry or lyophilized product by the manufacturer of the productor by the consumer during preparation.

The Bacillus coagulans spores survive storage (shelf-life), i.e., retainviability or the ability to germinate at physiological conditions (e.g.,ingestion), from about 12 days to about 2 years; from about 1 month toabout 18 months; from about 3 months to about 1 year; or from about 6months to about 9 months.

EXAMPLE 1: PREPARATION OF BACILLUS COAGULANS CULTURES

Bacillus coagulans Hammer bacteria (ATCC Accession No. 31284) wasinoculated and grown to a cell density of about 10⁸ to 10⁹ cells/ml innutrient broth containing 5 g Peptone, 3 g Meat extract, 10-30 mg MnSO₄,and 1,000 ml distilled water, adjusted to pH 7.0, using a standardairlift fermentation vessel at 30° C. The range of MnSO₄ acceptable forsporulation is 1 mg/l to 1 g/l. The vegetative cells can activelyreproduce up to 45° C., and the spores are stable up to 90° C. Afterfermentation, the B. coagulans bacterial cells or spores are collectedusing standard methods (e.g., filtration, centrifugation) and thecollected cells and spores can be lyophilized, spray-dried, air-dried orfrozen.

A typical yield from the above culture is in the range of about 10⁹ to10¹⁰ viable spores and more typically about 100 to 150 billioncells/spores per gram before drying. Spores maintain at least 90%viability after drying when stored at room temperature for up to tenyears, and thus the effective shelf life of a composition containing B.coagulans Hammer spores at room temperature is about 10 years.

EXAMPLE 2: PREPARATION OF BACILLUS COAGULANS SPORES

A culture of dried B. coagulans spores was prepared as follows. Tenmillion spores were inoculated into a one liter culture containing 24 gpotato dextrose broth, 10 g of enzymic-digest of poultry and fishtissue, 5 g of FOS and 10 g MnSO₄. The culture was maintained for 72hours under a high oxygen environment at 37° C. to produce culturehaving about 150 billion cells per gram of culture. Thereafter, theculture was filtered to remove culture medium liquid, and the bacterialpellet was resuspended in water and freeze-dried. The freeze-driedpowder is then ground to a fine powder using standard good manufacturingpractice (GMP). A dried composition that comprises at least about 75%(e.g., 80%, 85%, or 90%) spores is then mixed with protein powder.

EXAMPLE 3: BACILLUS COAGULANS SPORES SURVIVE IN THE GASTRIC ENVIRONMENT

This study was performed in order to determine the survivability rate ofBacillus coagulans spores as they pass through the stomach. Samples ofBacillus coagulans spores were subjected to a simulated gastricenvironment for varying lengths of time in order to attain theirsurvivability rate. First, a homogeneous sample of raw material Bacilluscoagulans of at least 12 grams was prepared. Saline solution at pH 1 wasprepared using 3N HCl (150 mls each into six 250 ml media bottles) andsterilized. Additional saline solutions with pH 2 and 3 were preparedsimilarly, resulting in 6 sterile 250 ml bottles, each containing 150 mlpH adjusted saline. Six sterile 250 ml media bottles each containing 150ml normal saline solution were prepared and sterilized. Phosphate buffer(˜400 ml) was prepared at pH 7.2. Test tubes (24) were prepared andsterilized, each containing 9 ml of phosphate buffer pH 7.2. Test tubes(120) were prepared, each containing 9 ml of normal saline. GYE(glucose-yeast extract) agar medium was prepared and sterilized andcooled to 45° C. in a water bath. Samples (24) of raw material wereweighed, each 500 milligrams (theoretically equivalent to 10 billionspores). The samples were added to media bottles at 37° C. and incubatedhalf for 20 minutes the other half for 120 minutes. After 20 and 120minutes incubation, respectively, the samples were mixed to uniformityand pipet 1 ml into 9 ml of sterile phosphate buffer pH 7.2. After all12 samples from each time point were placed into test tubes containingsterile phosphate buffer, serial dilutions were made until 6 tubes hadbeen used for each sample. The final dilution for the final two testtubes were 3×10⁷ and 3×10⁸, which gave a count of roughly 300 and 30CFU, respectively. The final 2 test tubes from each sample were placedinto 70° C. water bath for 30 minutes. After 30 minutes, they werecooled immediately to 45° C. Three sterile petri plates per tube wereset out. 1.0 ml from the heat-treated tube was added into each petriplate, then 15 ml of sterile molten GYE Agar medium (at 45° C.) waspoured into each of the petri plates and mixed thoroughly. Whensolidified, the plates were incubated in an inverted position for 48hours at 40° C. The individual colonies were counted. Results wereexpressed as CFU per gram as shown in Table 1 below. 1.0E+10 =1×10¹⁰.

TABLE 1 20 Minutes 120 Minutes Incubation Incubation Spore Count, SporeCount, Sample CFU/gram CFU/gram Normal Saline - A 1.90E+10 1.88E+10Normal Saline - B 2.12E+10 2.00E+10 Normal Saline - C 1.64E+10 2.06E+10Average 1.89E+10 1.98E+10 Saline pH 1.0 - D 2.08E+09 5.98E+07 Saline pH1.0 - E 1.47E+09 0.00E+00 Saline pH 1.0 - F 3.59E+09 0.00E+00 Average2.38E+09 1.99E+07 Saline pH 2.0 - G 3.63E+09 3.46E+09 Saline pH 2.0 - H4.47E+09 2.48E+09 Saline pH 2.0 - I 3.58E+09 2.82E+09 Average 3.89E+092.92E+09 Saline pH 3.0 - J 1.65E+10 1.13E+10 Saline pH 3.0 - K 1.35E+101.11E+10 Saline pH 3.0 - L 1.80E+10 1.39E+10 Average 1.60E+10 1.21E+10

EXAMPLE 4: THE EFFECTS OF A PROBIOTIC ON THE ABSORPTION OF PROTEIN ANDMICRONUTRIENTS IN HEALTHY MALES

The effects of the probiotic GANEDEN BC-30™ (Bacillus coagulans ATCCDesignation Number PTA-6086) on protein and micronutrient absorption isevaluated in healthy males following a 14-day period. The protocoloutlined below determines the effect of a probiotic product (GANEDENBC-30™ (Bacillus coagulans ATCC Designation Number PTA-6086) incombination with protein as compared to protein in the absence ofBacillus coagulans on protein absorption, both rate of absorption(t_(Max)) and overall absorption (from area under the curve (AUC_(0-4h))and C_(MAX)), as measured by changes in blood amino acid levels over afour hour test period. The protocol outlined below is also designed todetermine the effect of a probiotic product (GANEDEN BC-30™ (Bacilluscoagulans ATCC Designation Number PTA-6086) in combination with proteinas compared to protein in the absence of Bacillus coagulans on vitaminand mineral absorption, both rate of absorption (t_(Max)) and overallabsorption (from AUC_(0-4h) and C_(Max)), as measured by changes inblood levels of select vitamins (vitamins B6, B12 and C) and minerals(calcium, iron and zinc) over a four hour test period. The study alsodetermines the effect of a probiotic product (GANEDEN BC-30™ (Bacilluscoagulans ATCC Designation Number PTA-6086) in combination with proteinas compared to protein in the absence of Bacillus coagulans ongastrointestinal (Gl)-specific symptoms using a GI questionnaire after14 days of treatment.

The period of evaluation lasts approximately four weeks with subjectsattending a screening/randomization visit and two follow-up test visits.Eligible subjects take GANEDEN BC-30™ (Bacillus coagulans ATCCDesignation Number PTA-6086) plus protein and protein alone, each for 14days, in random order. After each 14-day period, there is a test visitat which subjects take the test product (probiotic plus protein orprotein alone) and a multiple vitamin and mineral supplement. Blood iscollected at baseline (prior to the test product) and at 1, 2, 3 and 4hours following product administration. Levels of various amino acids,vitamins and minerals are measured so that the effects of the probioticon protein and micronutrient absorption are determined. The probioticincreases the absorption of the protein and micronutrients by increasingthe efficiency of the gut by balancing the intestinal flora or byincreasing the acidity of the stomach for more efficient breakdown ofthe nutrients.

Although the study population is comprised of generally healthy adultsthat do not have GI disorders or symptoms, there may be beneficialeffects in GI health with the administration of GANEDEN BC-30™ (Bacilluscoagulans ATCC Designation Number PTA-6086). A six item questionnaire isused to assess changes in GI health (e.g. abdominal pain, bloating andgas) with lower incidence indicating positive effects with regard to GIhealth.

Study Design

The study described herein is a prospective, randomized, double blind,placebo controlled, crossover clinical trial. A total of 10 malesubjects are enrolled, with each subject receiving the two testproducts, in a randomly-assigned sequence. Each subject serves as hisown control.

Study Population:

The study population includes 10 male subjects, aged 21 to 60 years,that do not have any gastrointestinal disease or inflammatory bowelcondition, and who are not taking on a regular basis any prescription orover-the counter medications for any gastrointestinal problems.

Test Products:

Product 1—GANEDEN BC-30™ (Bacillus coagulans strain GBI-30, ATCCDesignation Number PTA-6086) plus Protein.Product 2—Protein (placebo; no probiotic).

Duration of Study

Excluding the screening visit, each subject completing the studyparticipates for approximately four weeks, ±three days.

Efficacy Assessments:

Efficacy variables consist of protein absorption [rate of absorption(t_(Max)) and overall absorption (AUC_(0-4h) and C_(Max)) of blood aminoacids], vitamin absorption [rate of absorption (t_(Max)) and overallabsorption (AUC_(0-4h) and C_(Max)) of vitamins B6, B12 and C], mineralabsorption [rate of absorption (t_(Max)) and overall absorption(AUC_(0-4h) and C_(Max)) of minerals calcium, iron and zinc], and GIquestionnaire scores (abdominal pain, abdominal bloating, gas, bowelmovements, gurgling noises and overall well-being).

Efficacy endpoints generally consist of changes in efficacy variablesfrom baseline to four hours post product administration following 14days of product use. These changes (endpoints) are then be comparedbetween the two products.

Statistical Methods:

For each efficacy endpoint (amino acid, vitamin, and mineral bloodconcentration), the following non-compartmental pharmacokinetic (PK)parameters are calculated:

C_(Max) The maximum concentration observed during the four post-dosesamples;t_(Max) the time at which the maximum concentration was observed;AUC_(0-4h) the area under the concentration-vs-time curve above thebaseline (time 0) concentration, integrated from time 0 to 4 hourspost-dose, by trapezoidal-rule quadrature.

All safety and efficacy variables are summarized by time point and byproduct. Numerical variables are presented as mean, standard deviation,count, median, and range (minimum to maximum value). Changes from theappropriate baseline are summarized and presented in the same way.Numerical variables and their changes from baseline are displayedgraphically, as plots of mean value vs. time, with separate lines forthe two products (probiotic vs. placebo). Categorical variables arepresented as tabulations of counts and percentages of totals.

For each continuous variable, the mean change from baseline to eachsubsequent time point are tested for nominal significance by the pairedStudent t test, or by the non-parametric Wilcoxon test if non-normallydistributed. The mean differences in the variable, or in the change inthat variable from baseline, between the different products are testedfor nominal significance by the paired Student t test or by thenon-parametric Wilcoxon test if non-normally distributed. For eachcategorical variable, difference in the distribution of categoriesbetween the different product groups are tested for nominal significanceby the Fisher Exact test if possible, or by the Chi-Square test ifnecessary.

Adverse events (AEs) are listed, Medical Dictionary for RegulatoryActivities (MedDRA) encoded, grouped by general type of event(gastrointestinal, neurologic, cardiac, etc.), and cross-tabulated byevent type and product. Differences in AE patterns between the twoproducts are tested by the Fisher Exact test. Subjective remarks arecategorized to the extent possible, and analyzed for pattern differencesbetween the two products in the same way as AE's.

The pharmacokinetic endpoints (C_(Max), t_(Max), and AUC_(0-4h)) after14 days of product use are tested for significance between the twoproducts by a repeated-measures analysis of variance (RM-ANOVA) for eachendpoint, where the value of the variable at each point in time is therepeated dependent variable, and the product identification (GANEDENBC-30™, Bacillus coagulans ATCC Designation Number PTA-6086+Protein orPlacebo) is the main factor . If the efficacy variables are not normallydistributed, and cannot be normalized by logarithmic or othertransformations, then between-product testing are carried out using theWilcoxon signed-ranks test.

Assuming 15% attrition during the course of the study, there are about 8or 9 analyzable subjects remaining in the per-protocol population. Tohave 80% power to obtain significance (p<0.05) when testing a changeover time (over 14 days of product use) within a product group, the meanchange is about equal to about 15% larger than the standard deviation ofthe changes (a “1.15-sigma” effect size). To show that there was asignificant difference between the two products, the mean difference isabout 65% larger than the within-group variability of the endpoint (a“1.65-sigma” effect size).

Each efficacy endpoint is considered an independent question ofinterest, and is tested independently at the 0.05 alpha level (p≤0.05required for a conclusion of statistical significance). Multiple testingis taken into account when the results of the efficacy analyses areinterpreted by the statistician in the Final Statistical Report.

Study Population Inclusion Criteria

1. Male subjects, aged 21 to 60 years

2. Subject is able to understand and sign the informed consent toparticipate in the study.

3. Subject is willing and able to comply with the protocol including:

-   -   a. Attending three visits;    -   b. Refraining from eating any yogurt or lacto-fermented        beverages during the study;    -   c. Refraining from using any dietary supplements or nutritionals        during the study;    -   d. Not taking any new vitamin and/or mineral supplements until        after study completion and refraining from taking any vitamin        and/or minerals supplements for 24 hours prior to the test        visits.

Exclusion Criteria

1. Subject has any of the following medical conditions:

-   -   a. active heart disease    -   b. uncontrolled high blood pressure (≥140/90 mmHg)    -   c. renal or hepatic impairment/disease    -   d. Type I or II diabetes    -   e. bipolar disorder    -   f. Parkinson's disease    -   g. unstable thyroid disease    -   h. immune disorder (such as human immune deficiency virus        (HIV)/acquired immune deficiency syndrome (AIDS))    -   i. psychiatric disorders (hospitalized within the past one year)    -   j. any medical condition deemed exclusionary by the Principal        Investigator (PI)

2. Subject has a history of cancer (except localized skin cancer withoutmetastases) within five years prior to screening.

3. Subject has a history of or currently has any gastrointestinaldisease or disorder or any inflammatory bowel condition such as Crohn'sdisease, short bowel, ulcerative colitis, or Irritable Bowel Syndrome(IBS).

4. Subject is lactose intolerant (self-professed or diagnosed).

5. Subject has had any stomach or intestinal surgery (i.e. gastricbypass).

6. Subject takes on a regular basis (defined as two or more times perweek) any prescription or over-the counter medications for diarrhea,constipation, heartburn or any other gastrointestinal problems.

7. Subject is currently taking laxatives or has taken laxatives withinthe 30 days prior to screening/enrollment.

8. Subject is currently taking antibiotics (or any drug thatsignificantly interferes with bacterial flora) or has taken antibioticswithin the 60 days prior to screening/enrollment.

9. Subject is currently taking or has used in the past 30 daysprobiotics/prebiotics (including yogurt and lacto-fermented beverages)or any digestive enzymes [prescription or over-the-counter (OTC)].Thirty-day washout allowed.

10. Subject is on an unstable dose of medication (defined as fewer than90 days at the same dose).

-   -   a. Anti-hypertensives and anti-hyperlipidemic medications ok if        stable dose.

11. Subject is currently taking any medication deemed exclusionary byPI.

12. Subject has an allergy to soy, milk, chocolate or any of theingredients in the test product.

13. Subject has a history of drug or alcohol abuse in the past 12months.

14. Subject has begun/stopped smoking ≤6 months ago OR has plans tobegin/quit smoking.

15. Subject has any condition or abnormality that, in the opinion of theinvestigator, would compromise the safety of the subject or the qualityof the study data.

16. Subject is participating or has participated in another researchstudy within 30 days prior to the screening visit.

Concomitant Medications and Other Substances Permitted During the Study

Subjects may take the following substances during their participation inthis study:

-   -   Vitamins and/or minerals subject was taking prior to starting        the study (same frequency as prior to study start). However,        subjects cannot take any vitamins and/or minerals for the 24        hours prior to the test visits. If the subject was not        previously taking vitamins and/or minerals, he is asked to not        start taking any during the study.    -   Thyroid hormone replacement therapy, if drug and dose are        unchanged for at least 90 days before screening and throughout        the study.    -   Antihypertensive and antihyperlipidemic medications, if drug and        dose are unchanged for at least 90 days before screening and        throughout the study.

Substances Not Permitted During the Study

Subjects may not take the following substances for at least 60 daysprior to the screening/randomization visit (visit 1) or throughout thestudy. Any subject taking these substances during the study is evaluatedby the PI.

-   -   All oral antibiotics including: Amoxil®, Trimox® (amoxicillin),        Cipro® (ciprofloxacin), Flagyl® (metronidazole), Doxy-100®        (doxycycline) and Bactrim® Double Strength        (trimethoprim-sulfamethoxazole).

Any medications that significantly interfere with bacterial floraSubjects may not take the following substances for at least 30 daysprior to the screening/randomization visit (visit 1) or throughout thestudy. Any subject taking these substances during the study is evaluatedby the PI.

-   -   Laxatives: psyllium husk (Metamucil), methylcellulose        (Citrucel), polycarbophil, docusate (Colace, Diocto), mineral        oil, sodium phosphate (and variants), magnesium citrate,        magnesium hydroxide (Milk of magnesia), magnesium sulfate (which        is Epsom salt), glycerin suppositories, sorbitol, lactulose, and        polyethylene glycol (PEG)    -   Digestive enzymes: Cotazym, Creon, Pancrease, Digex, Dygase,        Gastrinex, Hi-Vegi-Lip, Ku-Zyme, Kutrase, Lactaid, Lapase,        Lipram, Palcaps 10, Pan-2400, Pancreatin, Pancrecarb,        Pangestyme, Panocaps, Panokase, Sucraid, SureLac, Ultrase,        Viokase, Zenpep    -   All dietary and herbal supplements including:        -   Probiotic products (including fortified foods): Acidophilus,            Bacid, Flora-Q, Florastor, Novaflor, RisaQuad,            Superdophilus, GANEDEN BC-30™ (Bacillus coagulans ATCC            Designation Number PTO-6086) LAFTIB94, LAFTIL10, LAFTI L26,            Bifiene, Align, Howaru Bifido, ProBactrix, Mutaflor,            Actimel/DanActive, Cultura, Yakult, Lactobacillus fortis,            GoodBelly/ProViva/TuZen, LGG, Vifit, Verum, DiarSafe, Bion            Flore Intime, Jarrow, Fem-Dophilus, Florajen3, Bio-K,            CL1285, A′Biotica;        -   Prebiotic products (including fortified foods): lactulose,            lactitol oligofructose, inulin, galacto-oligosaccharides,            tagatose, isomaltooligosaccharides, polydextrose,            maltodextrin, fructo-oligosaccharides, arabinogalactan,            polyols-lactulose;        -   Those supplements purported to improve GI health including            dandelion, devil's claw, feverfew, ginger, goldenseal, lemon            balm, peppermint, roman chamomile, turmeric, valerian,            yarrow, mastic gum, dill, caraway, anise, cumin.

Diet

Subjects are required to fast (no food or beverage other than water, nocaffeine) for eight hours prior to visits 2 and 3. Subjects are asked torefrain from alcohol for the 24 hours prior to visits 2 and 3. Subjectsare not allowed to eat yogurt or to drink lacto-fermented beveragesthroughout the study period. Examples of lacto-fermented beveragesinclude kefir (water and dairy), kambucha, kvass, cider and ginger ale.

Subjects are not allowed to use protein powders (e.g. whey, soy, hemp,pea, rice) while participating in the study.

Subjects are not allowed to eat/drink foods and beverages high invitamin C for 24 hours prior to the test visits. Examples of foods andbeverages high in vitamin C include citrus fruits (such as oranges andgrapefruit) and their juices, red and green pepper, kiwifruit, broccoli,strawberries, cantaloupe, baked potatoes, and tomatoes.

Other than this, subjects are allowed to continue their normal diet.

Exercise

Subjects are asked to refrain from exercise for the 24 hours prior tovisits 2 and 3. Subjects are allowed to continue their usual exerciseroutine. Product Ingredients

The following lists of ingredients are provided.

Product 1: GANEDEN BC-30™ (Bacillus coagulans ATCC Designation NumberPTA-6086)+Protein

1 billion CFU GANEDEN BC-30™ (Bacillus coagulans ATCC Designation NumberPTA-6086) and 23 g of whey protein (whey protein isolate (WPI)>90) in 30g of powder, chocolate flavor.

Product 2: Protein (placebo)

23 g of whey protein (WPI >90) in 30 g of powder, chocolate flavor.

Nutrition Facts for the Whey Protein Powder

Serving Size: 1 Packet Amount Per Serving Calories 130 Calories from fat25 % Daily Value* Total Fat 2.5 g 4% Saturated Fat 1 g 5% Trans Fat 0 gCholesterol 65 mg 22%  Sodium 50 mg 2% Potassium 170 mg 5% TotalCarbohydrate 3 g 1% Dietary Fiber 1 g 4% Sugars 1 g Protein 23 g 46% Vitamin A 0% Vitamin C 0% Calcium 10%  Iron <2%  *Percent Daily Valuesare based on a 2,000 calorie diet.Ingredients: Whey Protein Concentrate (Whey Protein Concentrate, SoyLecithin), Whey Protein Isolate, Cocoa Powder (Processed with Alkali),Natural & Artificial Flavors, Acesulfame Potassium and Sucralose.

At the test visits, subjects are provided a multiple vitamin withmineral supplement. The supplement that is used is the One A Day® Men'sHealth Formula.

The Ingredients in the One A Day Men's Health Formula

Calcium Carbonate, Magnesium Oxide, Microcrystalline Cellulose, AscorbicAcid, Croscarmellose Sodium, Gelatin, Maltodextrin; Less than 2% of:Beta-Carotene, Biotin, Cholecalciferol, Chromium Chloride, Crospovidone,Cupric Oxide, Cyanocobalamin, D-Calcium Pantothenate,dl-Alpha-Tocopheryl Acetate, Folic Acid, Hydroxypropyl Methylcellulose,Lycopene, Manganese Sulfate, Niacinamide, Phytonadione, PolyethyleneGlycol, Pyridoxine Hydrochloride, Riboflavin, Silicon Dioxide, SodiumSelenate, Soybean Oil, Starch, Stearic Acid, Thiamine Mononitrate,Triacetin, Vitamin A Acetate and Zinc Oxide.

Product Dosing

Product administration instructions: Subjects are instructed to take onepacket per day, at approximately the same time of day.

Preparation instructions: Mix one packet with six to eight ounces ofcold water, milk or subject's preferred beverage for 20 to 30 seconds ina shaker or blender. Subjects are instructed to start taking the firststudy product the day after visit 1. Subjects are instructed to not takethe study product prior to coming in for visits 2 and 3 (i.e. not takestudy product the morning of visits 2 and 3). Subjects are required totake the study product on site for visits 2 and 3. Subjects areinstructed to start the second study product the day after visit 2.Subjects cannot take anything the evening of visit 2. Subjects areinstructed to bring the remaining product to all visits.

Product Administration Instructions

Take one packet per day, at approximately the same time of day; mix with6 to 8 ounces of water, milk or favorite beverage and shake/blend for 20to 30 seconds.

The product is packaged so that the subject is provided only thequantity needed for the time between visits (14 days) plus product tocover the window (additional three days of product).

Product Compliance

Compliance is measured via the packet counting method. By documentingthe number of calendar days between visits and the number of packetsthat should have been taken, compliance is calculated. The subject'scompliance is recorded (as a % of prescribed amount) for each productand this compliance % is used to establish the subject's eligibility forinclusion in the populations (Safety, Intent-to-Treat, and/orPer-Protocol) used for analysis of the study's results, in accordancewith the criteria specified below.

For visits 2 and 3, product administration are performed on site.Documentation is done in each subject's source confirming productadministration.

Randomization

The consulting statistician develops a randomization schedule and createa randomization log for use at the investigative site. To minimize theeffect of treatment sequence (carry-over, training, fatigue,seasonality, etc.) on safety and efficacy endpoints, the active product(A) and placebo (B) are administered to each subject in one of the twopossible sequences: A-B or B-A. Block-2 randomization is used, so thatthe two possible product sequences are shuffled into random order forassignment for the first two subjects, then shuffled again into adifferent random permutation for assignment to the next two subjects,and so on for the remaining subjects to be enrolled (plus additionalspare product).

Study Procedures Informed Consent

An informed consent form (IGF) is written in accordance with establishedcriteria of the Institutional Review Board (IRB) and the appropriatefederal regulations (eg, 21 CFR Parts 50 and 56) to describe the studyplan, procedures, and risks. The investigator and the IRB must allapprove the IGF and any IGF amendments or administrative changes beforethey are used.

Investigators ensure that each subject is clearly and fully informed ofthe purpose, potential risks, and requirements of study participation.Written informed consent must be obtained from each subject beforeperforming any screening or other study procedures.

Medical History

A Medical History is performed at the screening visit to collectinformation on past and current medical conditions, surgical history,allergy information, and concomitant or recently taken (in the past 90days) medications including over the counter (OTC) non-prescriptionproducts, nutritional supplements, herbals, and investigationalproducts.

Physical Examination

Subjects undergo a full physical examination at screening fordetermining eligibility.

Vital Signs

At each visit, subjects have blood pressure and heart rate measured.

The following guidelines are used for the blood pressure and heart rate:subjects sit in a chair, resting, for at least five minutes prior totaking the measurements. Measurements are done with subjects in a seatedposition and the subject's left arm is used.

Study Questionnaires

-   -   Dichotomous Questionnaire—the following is administered at        visits 2 and 3. Each subject is asked:        -   Did you:            -   Not eat or drink any foods or beverages other than water                or have caffeine for the eight hours prior to the visit?            -   Eat or drink any foods or beverages high in vitamin C in                the past 24 hours?            -   Drink any alcoholic beverages in the past 24 hours?            -   Take any vitamin and/or mineral supplements in the past                24 hours?            -   Exercise in the past 24 hours?            -   Use any new vitamin and/or mineral supplements since the                screening visit?            -   Use any protein powder other than the study product                since the screening visit?            -   Use any dietary supplements, any probiotics, or                prebiotics since 30 days prior to the screening visit?            -   Eat or drink any yogurt or lacto-fermented beverages                since 30 days prior to the screening visit?            -   Did you take the study product today?        -   If subject answers “yes” to any of the above questions, the            subject is discontinued as per PI discretion and/or a            protocol deviation is recorded.    -   GI Questionnaire—a six-part questionnaire regarding general        well-being and gastrointestinal symptoms is administered at        visits 1, 2 and 3. Subjects are asked, “how often in the past        two weeks have you” experienced various GI symptoms with answer        choices of “all of the time, most of the time, sometimes, a        little and never”. Subjects are asked to rate how they feel        overall with answer choices of “better, same and worse”. See,        e.g., Worthley et al, 2009 Am J Clin Nutr, 90: 578-586. For        example, the following questions are asked:    -   1) How often in the past week have you had pain in the abdomen?    -   2) How often in the past week have you experienced abdominal        bloating?    -   3) How often in the past week have you been troubled by        excessive passage of gas through the anus?    -   4) How often in the past week have you been troubled by frequent        of loose bowel movements?    -   5) How often in the past week have you been troubled by        excessive gurgling noises from the abdomen?    -   6) In comparison to how you felt at the start of the study, over        the past week, have you felt better, worse or the same?

Protein and Micronutrient Absorption Test

The following test is performed at visits 2 and 3 to assess the effectsof the products on protein, vitamin and mineral absorption.

The following is performed to standardize the testing conditions:

The test is performed after an eight hour fast;

Subjects are not physically active during the test;

No smoking is allowed during the test.

Test Procedure:

-   -   Collect the baseline blood sample (0 hours) for the evaluation        of the baseline levels of various amino acids and select        vitamins and minerals.    -   Provide subject with the multiple vitamin with mineral        supplement (can be taken with water or the study product).    -   Provide subject with the study product which is to be consumed        within 10 minutes.    -   Timing of the test is from when the subject begins to drink the        study product.    -   Collect blood samples at 1, 2, 3, and 4 hours for the evaluation        of the absorption of the amino acids, vitamins and minerals.

During the Time Between Blood Draws:

-   -   Subjects are primarily sitting.    -   Subjects are allowed to walk around the site to use the rest        room and to stretch but are not allowed to do any additional        physical activity.    -   Subjects are allowed to do restful activities such as read,        listen to music with earphones, watch television, work on the        computer or rest/sleep.    -   Subjects are not allowed to eat or drink anything other than        water.

Descriptive Summarization

For each efficacy endpoint (amino acid, vitamin, and mineral bloodconcentration), the following non-compartmental pharmacokinetic (PK)parameters is calculated:

-   -   C_(Max) The maximum concentration observed during the four        post-dose samples;    -   t_(Max) the time at which the maximum concentration was        observed;    -   AUC_(0-4h) the area under the concentration-vs-time curve above        the baseline (time 0) concentration, integrated from time 0 to 4        hours post-dose, by trapezoidal-rule quadrature.

All safety and efficacy variables are summarized by time point and byproduct. Numerical variables are presented as mean, standard deviation,count, median, and range (minimum to maximum value). Changes from theappropriate baseline are summarized and presented in the same way.Numerical variables and their changes from baseline are displayedgraphically, as plots of mean value vs. time, with separate lines forthe two products (probiotic vs. placebo). The graphs may containvertical error-bars around the mean values, indicated standard errors ofthe mean, if, in the opinion of the statistician, this would make thegraphs more informative. Categorical variables are presented astabulations of counts and percentages of totals.

For each continuous variable, the mean change from baseline to eachsubsequent time point are tested for nominal significance by the pairedStudent t test, or by the non-parametric Wilcoxon test if non-normallydistributed.

For each continuous variable at each time point, the mean differences inthe variable, or in the change in that variable from baseline, betweenthe different products are tested for nominal significance by the pairedStudent t test or by the non-parametric Wilcoxon test if non-normallydistributed.

For each categorical variable, difference in the distribution ofcategories between the different product groups are tested for nominalsignificance by the Fisher Exact test if possible, or by the Chi-Squaretest if necessary.

All p-values appearing in these summarizations is considereddescriptive, not inferential. No final statistical conclusions are drawnfrom them, but they are referred to in the interpretation of thechanges.

Statistical Analysis Safety and Efficacy Variables and Endpoints

Safety variables consist of adverse events and subjective remarks.

Safety endpoints consist of the changes in the safety variables listedabove with each product following 14 days of treatment.

There is no formal safety objective.

Efficacy variables consist of protein absorption [rate of absorption(t_(Max)) and overall absorption (AUC_(0-4h) and C_(Max)) of blood aminoacids], vitamin absorption [rate of absorption (t_(Max)) and overallabsorption (AUC_(0-4h) and C_(Max)) of vitamins B6, B12 and C], mineralabsorption [rate of absorption (t_(Max)) and overall absorption(AUC_(0-4h) and C_(Max)) of minerals calcium, iron and zinc], and GIquestionnaire scores (abdominal pain, abdominal bloating, gas, bowelmovements, gurgling noises and overall well-being).

Efficacy endpoints generally consist of changes in efficacy variablesfrom baseline to four hours post product administration following 14days of product use. These changes (endpoints) are then compared betweenthe two products.

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. Genbank and NCBI submissions indicated byaccession number cited herein are hereby incorporated by reference. Allother published references, documents, manuscripts and scientificliterature cited herein are hereby incorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A sports nutrition composition comprising a protein source and anisolated Bacillus coagulans spore, wherein said composition comprises atleast 50% protein.
 2. The composition of claim 1, wherein saidcomposition comprises at least 75% protein.
 3. The composition of claim1, wherein said isolated Bacillus coagulans is Bacillus coagulans hammerstrain Accession No. ATCC
 31284. 4. The composition of claim 1, whereinsaid isolated Bacillus coagulans is selected from the group consistingof GBI-30 strain (ATCC Designation Number PTA-6086), GBI-20 strain (ATCCDesignation Number PTA-6085), and GBI-40 strain (ATCC Designation NumberPTA- 6087).
 5. The composition of claim 1, wherein said compositioncomprises about 1×10⁶ to 1×10¹⁴ colony forming units (CFU) of Bacilluscoagulans per unit dose.
 6. The composition of claim 1, wherein saidcomposition comprises about 20 grams of said protein to about 50 gramsof said protein per unit dose.
 7. The composition of claim 1, whereinsaid protein comprises whey protein, brown rice protein, casein milkprotein, egg albumin, and whey protein hydrolysate.
 8. The compositionof claim 1, wherein said composition comprises approximately 1 billionCFU Bacillus coagulans.
 9. The composition of claim 8, wherein saidprotein is soy protein, whey protein, rice protein, hemp seed protein,or casein protein.
 10. The composition of claim 8, wherein said proteincomprises an amino acid selected from the group consisting ofisoleucine, alanine, leucine, arginine, lysine, aspartate, asparticacid, methionine, cysteine, phenylalanine, threonine, tryptophan,glycine, valine, proline, histidine, serine, tyrosine, asparagine,selenocysteine, pyrrolysine, glutamate, glutamic acid, and glutamine.11. The composition of claim 8, further comprising creatine, calcium,sodium caseinate, whey peptides, or lactoferrin.
 12. The composition ofclaim 1, wherein said sports nutrition composition is selected from thegroup consisting of creatine, calcium, sodium caseinate, whey peptides,and lactoferrin.
 13. The composition of claim 1, wherein saidcomposition does not comprise an sugar.
 14. The composition of claim 1,wherein said composition does not comprise gluten.
 15. The compositionof claim 1, wherein said composition does not comprise aspartame. 16.The composition of claim 1, wherein said composition does not compriseartificial coloring.
 17. The composition of claim 1, wherein saidcomposition is in the form of a protein powder, a ready to drink proteinshake, a protein bar, a protein bite, or a protein gel.
 18. Thecomposition of claim 1, wherein said composition comprises a weightgainer.
 19. A composition comprising a dry mix for sports nutritioncomposition comprising a sports nutrition composition and an isolatedBacillus coagulans spore.
 20. The composition of claim 1, wherein saidprotein comprises whey protein.
 21. The composition of claim 19, whereinsaid isolated Bacillus coagulans is selected from the group consistingof GBI-30 strain (ATCC Designation Number PTA-6086), GBI-20 strain (ATCCDesignation Number PTA-6085), and GBI-40 strain (ATCC Designation NumberPTA- 6087).
 22. The composition of claim 19, wherein said sportsnutrition composition comprises protein selected from the groupconsisting of soy protein, whey protein, rice protein, hemp seedprotein, or casein protein.
 23. The composition of claim 22, whereinsaid protein comprises an amino acid selected from the group consistingof isoleucine, alanine, leucine, arginine, lysine, aspartate, asparticacid, methionine, cysteine, phenylalanine, threonine, tryptophan,glycine, valine, proline, histidine, serine, tyrosine, asparagine,selenocysteine, pyrrolysine, glutamate, glutamic acid, and glutamine.24. The composition of claim 19, wherein said sports nutritioncomposition is selected from the group consisting of creatine, calcium,sodium caseinate, whey peptides, and lactoferrin. 25-33. (canceled)